Project Summary. Ongoing studies of the mechanistic enzymology of complex polyketide natural product biosynthesis will be extended, with focus on the biosynthesis of the macrolide antibiotics erythromycin, methymycin, picromycin, and tylosin, the protein phosphatase inhibitor fostriecin, and the polyether antibiotic nanchangmycin. Each of these metabolites is assembled by closely related, exceptionally large, multifunctional, modular proteins known as polyketide synthases (PKSs). A combination of chemical, enzymological, and protein molecular engineering techniques is being used to elucidate the molecular basis for the programming and mechanism of the complex series of reactions by which these polyketides are assembled. The emphasis is on the mechanism, stereochemistry, and specificity of multistep, enzyme- catalyzed transformations leading to the formation of these medicinally important natural products. 1) The enzymology and mechanism of double bond formation in complex polyketide biosynthesis will be probed by expression in E. coli of individual modules of several PKS proteins, each responsible for a single round of polyketide chain elongation and functional group modification. Selected mutants will be generated in order to establish the structure and stereochemistry of otherwise transient chain elongation intermediates. 2) Pair wise incubations of ketosynthase and ketoreductase domains from several PKS modules will be used to determine the mechanistic role played by each domain in fixing the stereochemistry of methyl and hydroxyl substituents in the resultant polyketide products. The results of these studies will be not only be broadly applicable to the understanding of polyketide antibiotic biosynthesis will contribute valuable tools for the rational engineering of novel polyketides with potential antibiotic, antitumor, and other activities. In addition, the results obtained will provide fundamental insights into how catalysis and molecular recognition control both substrate specificity and product molecular diversity in Nature.